An atrial peptide with natriuretic and diuretic properties was first reported from rat atrial muscle in 1981. Since then a family of natriuretic hormone peptides (NP) with broad physiologic effects including vasodilation and inhibition of aldosterone secretion has been described. Atrial natriuretic factor (ANF), a 126 amino acid prohormone gives rise to four peptides: long acting natriuretic peptide (LANP, amino acids 1-30), vessel dilator (VD, residues 31-67), kaliuretic peptide (KP, residues 79-98) and atrial natriuretic peptide (ANP, residues 99-126, also referred to here as NP99-126) (Vesely, D L Cardiovasc Res, 2001 51:647-58). In addition, renal tubular cells produce urodilatin, a 32 amino acid peptide (residues 95-126 of ANF), which is released to circulation following differential processing of ANF (Forssmann et al. Cardiovasc Res, 2001, 51:450-62.). There is also a pro-brain natriutretic peptide (BNP) first discovered in porcine brain, which is analogous to ANP is found in circulation. The third type of natriuretic hormone, the C-type (CNP) comprises two peptides, 53 and 22 amino acids in length, which are produced by many cell types (Levin, E R et al. N Eng J Med, 1998, 339321-8). Of these peptides, the C-terminal pro-ANF, ANP, has been studied most extensively.
In keeping with the diversity of these NPs, there are three NP receptors (Misono, K S Mol Cell Biochem, 2002, 230(1-2):49-60; Tremblay, J et al. Mol Cell Biochem, 2002, 230(1-2):31-47). NPRa and NPRb, which are coupled to guanylyl cyclase, and the cGMP-independent receptor NPRc. ANP and BNP signal primarily through NPRa, which increases cGMP and activates cGMP-dependent protein kinase (PKG). PKG activation turns on the ion transport mechanism and activates specific transcription factors, which together affect a range of cellular activities including, cell growth and proliferation, apoptosis and inflammation. NPRC functions as a clearance receptor but also appears to signal phospholipase C activation and a decrease in adenylyl cyclase activity (Silberbach, M et al. Cell Signal, 2001 13:221-31). Numerous tissues of various organ systems including the lung express these receptors in diverse cells.
The NPs are produced in various tissues of the mucosa (lung, gastrointestinal and genitourinary systems), central nervous system and cardiovascular systems and released into the circulation. The signaling mechanisms underlying ANP's growth inhibitory effects are poorly understood, although a number of reports suggest that ANP affects signaling via activation of mitogen-activated protein kinases (Silberbach, M et al. Cell Signal, 2001 13:221-31). The potential effects may include inhibition of ERK activation of epidermal growth factor, PKG-induced uncoupling of Ras/Raf1 interaction, or induction of MKP-1, a MAPK phosphatase that inactivates signaling through a number of growth factors such as endothelin, EGF and FGF (Clark, A R J Endocrinol, 2003, 178: 5-12). ANP has been shown to mediate anti-inflammatory (Kiemer, A K and Vollmar J Biol Chem, 1998 273:134444-51) and cytoprotective (Kiemer, A K et al., J Immunol, 2000, 165:175-81; Sprenger, H et al., Immunobiology, 1991, 183:94-101) effects. It has been shown to decrease cytokine and stress stimulated activation of NFκB in various cell types, leading to a decrease in pro-inflammatory cytokine production (Kiemer, A K and Vollmar J Biol Chem, 1998 273:134444-51; Kiemer, A K et al., J Immunol, 2000, 165:175-81; Morita, R et al., J Immunol, 2003:170:5869-75). ANP can reduce tumor necrosis factor-α (TNF-α)-stimulated production of adhesion molecules in endothelium. (Kiemer, A K and Vollmar J Biol Chem, 1998 273:134444-51). It has also been shown to attenuate TNF-α-induced actin polymerization, through activation of MAPK phoshatase-1 (MKP-1) and inhibition of p38 activity, leading to decreased permeability (Clark, A R J Endocrinol, 2003, 178(1):5-12).
ANP stimulates migration of human neutrophils (Izumi, T et al. J Clin Invest, 2001, 108(2):203-21345), and inhibits nitric oxide (NO) and TNF-a production by murine macrophages (Vesely, D L et al. Chest, 1990, 97(6):1295-1298, Vesely, D L Am J Obstet Gynecol, 1991, 165(3):567-573). Human peripheral blood monocytes, however, do not express ANP receptors nor do they respond to ANP (Sprenger, H et al. Immunobiology, 1991, 183(1-2):94-101). The NP system, acting via cells of the innate immune system, modulates the immune response to antigens. Evidence to date suggests that it may augment allergic inflammation by acting on a number of cells in the lung (Kurihara, M et al. Biochem Biophys Res Commun, 1987, 149(3):1132-1140). The primary evidence supporting this notion is the finding that ANP acts via its receptor on dendritic cells to polarize these cells toward a Th2 phenotype, which is the hallmark of allergic immune response (Morita R et al. J Immunol, 2003, 170(12):5869-5875). In asthma, the production of inflammatory mediators secreted from resident epithelial cells and recruited immune cells results in airway hyperreactivity, which characterizes the late-phase airway response. Without intervention, this event leads to non-reversible airway remodeling (including sub-basement-membrane collagen deposition, smooth muscle hyperplasia and hypertrophy, and goblet cell hyperplasia), with subsequent airway narrowing and progression of the asthma.
A naturally occurring gene-silencing mechanism triggered by double-stranded RNA (dsRNA), designated as small interfering RNA (siRNA), has emerged as a very important tool to suppress or knock down gene expression in many systems. RNA interference is triggered by dsRNA that is cleaved by an RNAse-III-like enzyme, Dicer, into 21-25 nucleotide fragments with characteristic 5′ and 3′ termini (Provost, P. D. et al. Embo J, 2002, 21:5864). These siRNAs act as guides for a multi-protein complex, including a PAZ/PIWI domain containing the protein Argonaute2, that cleaves the target mRNA (Hammond, S. M. et al. Science, 2001, 293:1146-1150). These gene-silencing mechanisms are highly specific and potent and can potentially induce inhibition of gene expression throughout an organism. The short interference RNA (siRNA) approach has proven effective in silencing a number of genes of different viruses (Fire, A. Trends Genet., 1999, 15:358-363).
RNA interference (RNAi) is a polynucleotide sequence-specific, post-transcriptional gene silencing mechanism effected by double-stranded RNA that results in degradation of a specific messenger RNA (mRNA), thereby reducing the expression of a desired target polypeptide encoded by the mRNA (see, e.g., WO 99/32619; WO 01/75164; U.S. Pat. No. 6,506,559; Fire et al., Nature 391:806-11 (1998); Sharp, Genes Dev. 13:139-41 (1999); Elbashir et al. Nature 411:494-98 (2001); Harborth et al., J. Cell Sci. 114:4557-65 (2001)). RNAi is mediated by double-stranded polynucleotides, such as double-stranded RNA (dsRNA), having sequences that correspond to exonic sequences encoding portions of the polypeptides for which expression is compromised. RNAi reportedly is not effected by double-stranded RNA polynucleotides that share sequence identity with intronic or promoter sequences (Elbashir et al., 2001). RNAi pathways have been best characterized in Drosophila and Caenorhabditis elegans, but “small interfering RNA” (siRNA) polynucleotides that interfere with expression of specific polynucleotides in higher eukaryotes such as mammals (including humans) have also been investigated (e.g., Tuschl, 2001 Chembiochem. 2:239-245; Sharp, 2001 Genes Dev. 15:485; Bernstein et al., 2001 RNA 7:1509; Zamore, 2002 Science 296:1265; Plasterk, 2002 Science 296:1263; Zamore 2001 Nat. Struct. Biol. 8:746; Matzke et al., 2001 Science 293:1080; Scadden et al., 2001 EMBO Rep. 2:1107).
According to a current non-limiting model, the RNAi pathway is initiated by ATP-dependent, cleavage of long dsRNA into double-stranded fragments of about 18-27 (e.g., 19, 20, 21, 22, 23, 24, 25, 26, etc.) nucleotide base pairs in length, called small interfering RNAs (siRNAs) (see review by Hutvagner et al., Curr. Opin. Gen. Dev. 12:225-32 (2002); Elbashir et al., 2001; Nyknen et al., Cell 107:309-21 (2001); Zamore et al., Cell 101:25-33 (2000)). In Drosophila, an enzyme known as “Dicer” cleaves the longer double-stranded RNA into siRNAs; Dicer belongs to the RNase III family of dsRNA-specific endonucleases (WO 01/68836; Bernstein et al., Nature 409:363-66 (2001)). Further, according to this non-limiting model, the siRNA duplexes are incorporated into a protein complex, followed by ATP-dependent unwinding of the siRNA, which then generates an active RNA-induced silencing complex (RISC) (WO 01/68836). The complex recognizes and cleaves a target RNA that is complementary to the guide strand of the siRNA, thus interfering with expression of a specific protein (Hutvagner et al., supra).
In C. elegans and Drosophila, RNAi may be mediated by long double-stranded RNA polynucleotides (WO 99/32619; WO 01/75164; Fire et al., 1998; Clemens et al., Proc. Natl. Acad. Sci. USA 97:6499-6503 (2000); Kisielow et al., Biochem. J. 363:1-5 (2002); see also WO 01/92513 (RNAi-mediated silencing in yeast)). In mammalian cells, however, transfection with long dsRNA polynucleotides (i.e., greater than 30 base pairs) leads to activation of a non-specific sequence response that globally blocks the initiation of protein synthesis and causes mRNA degradation (Bass, Nature 411:428-29 (2001)). Transfection of human and other mammalian cells with double-stranded RNAs of about 18-27 nucleotide base pairs in length interferes in a sequence-specific manner with expression of particular polypeptides encoded by messenger RNAs (mRNA) containing corresponding nucleotide sequences (WO 01/75164; Elbashir et al., 2001; Elbashir et al., Genes Dev. 15:188-200 (2001)); Harborth et al., J. Cell Sci. 114:4557-65 (2001); Carthew et al., Curr. Opin. Cell Biol. 13:244-48 (2001); Mailand et al., Nature Cell Biol. Advance Online Publication (Mar. 18, 2002); Mailand et al. 2002 Nature Cell Biol. 4:317).
siRNA polynucleotides may offer certain advantages over other polynucleotides known in the art for use in sequence-specific alteration or modulation of gene expression to yield altered levels of an encoded polypeptide product. These advantages include lower effective siRNA polynucleotide concentrations, enhanced siRNA polynucleotide stability, and shorter siRNA polynucleotide oligonucleotide lengths relative to such other polynucleotides (e.g., antisense, ribozyme or triplex polynucleotides). By way of a brief background, “antisense” polynucleotides bind in a sequence-specific manner to target nucleic acids, such as mRNA or DNA, to prevent transcription of DNA or translation of the mRNA (see, e.g., U.S. Pat. No. 5,168,053; U.S. Pat. No. 5,190,931; U.S. Pat. No. 5,135,917; U.S. Pat. No. 5,087,617; see also, e.g., Clusel et al., 1993 Nucl. Acids Res. 21:3405-11, describing “dumbbell” antisense oligonucleotides). “Ribozyme” polynucleotides can be targeted to any RNA transcript and are capable of catalytically cleaving such transcripts, thus impairing translation of mRNA (see, e.g., U.S. Pat. No. 5,272,262; U.S. Pat. No. 5,144,019; and U.S. Pat. Nos. 5,168,053, 5,180,818, 5,116,742 and 5,093,246; U.S. Ser. No. 2002/193579). “Triplex” DNA molecules refers to single DNA strands that bind duplex DNA to form a colinear triplex molecule, thereby preventing transcription (see, e.g., U.S. Pat. No. 5,176,996, describing methods for making synthetic oligonucleotides that bind to target sites on duplex DNA). Such triple-stranded structures are unstable and form only transiently under physiological conditions. Because single-stranded polynucleotides do not readily diffuse into cells and are therefore susceptible to nuclease digestion, development of single-stranded DNA for antisense or triplex technologies often requires chemically modified nucleotides to improve stability and absorption by cells. siRNAs, by contrast, are readily taken up by intact cells, are effective at interfering with the expression of specific polynucleotides at concentrations that are several orders of magnitude lower than those required for either antisense or ribozyme polynucleotides, and do not require the use of chemically modified nucleotides.
Due to its advantages, RNAi has been applied as a target validation tool in research in vitro and as a potential strategy for in vivo target validation and therapeutic product development (Novina, C. D. and Sharp, P. A., Nature, 2004, 430:161-164; Lieberman, J. et al. Trends Mol. Med., 2003, 9(9):397-403). In vivo gene silencing with RNAi has been reported using viral vector delivery, liposomal delivery, and high-pressure, high-volume intravenous (i.v.) injection of synthetic iRNAs (Halder, J. et al. Clin. Cancer Res., 2006, 12(16):4916-4924; Landen, C. N. et al., Cancer Biol. Ther., 2006, 5(12):1708-1713; Scherr, M. et al. Oligonucleotides, 2003, 13:353-363; Song, E. et al. Nature Med., 2003, 347-351). In vivo gene silencing has been reported after local direct administration (intravitreal, intranasal, and intrathecal) of siRNAs to sequestered anatomical sites in various models of disease or injury, demonstrating the potential for delivery to organs such as the eye, lungs, and central nervous system (Reich, S. J. et al. Mol. Vis., 2003, 9:210-216; Zhang, X. et al. J. Biol. Chem., 2004, 279:10677-10684; Dorn, G. et al. Nucleic Acids Res., 2004, 32, e49; Tolentino, M. J. et al. Retina, 2004, 24:132-138). Silencing of endogenous genes by systemic administration of siRNAs has also been demonstrated (Zimmerman, T. S. et al., Nature, 2006, 441(7089):111-114; Soutschek, J. et al. Nature, 2004, 432:173-178).
The present inventors have demonstrated that, in contrast to prior knowledge that ANP decreases inflammatory mechanisms in the macrophages, ANP actually increases lung inflammation and this is caused by ANP-NPRA signaling. This signaling can be blocked by utilizing a small interference RNA (siRNA) approach, in which specific siRNAs targeted to NPRA can significantly decrease the inflammation. This results in amelioration of inflammation in allergic disease which may be caused by allergens and exacerbated by respiratory viral infections, pollutants, and smoke. Also, this may be beneficial in the amelioration of inflammation and tumorigenesis in cancers.